Abstract
BackgroundHigh-molecular-weight and pure DNA is crucial for high-quality results from 3rd generation DNA Analyzers and optical mapping technologies. Conventional nuclei isolation methods for preparing high-molecular-weight genomic DNA from plant tissues include the preparation of protoplasts or embedding nuclei in an agarose matrix with subsequent manipulations via electro-elution or pulsed-field gel electrophoresis.ResultsIn this method, plant nuclei are isolated by physically grinding tissues and reconstituting the intact nuclei in a unique Nuclear Isolation Buffer (NIB). The plastid DNAs are released from organelles and eliminated with an osmotic buffer by washing and centrifugation. The purified nuclei are then lysed and further cleaned by organic extraction, and the genomic DNA is precipitated with a high concentration of CTAB. The highly pure, high molecular weight gDNA is extracted from the nuclei, dissolved in a high pH buffer, allowing for stable long-term storage.ConclusionsThis method is unique and avoids the use of embedding in agarose, which dramatically reduces time (4–8 h versus days), complexity, and materials cost. This procedure can be used on essentially any plant species and tissue stage. Here we describe a case study and a simple method to rapidly prepare high molecular weight gDNA from Upland cotton, blackgrass, and strawberry suitable for single-molecule sequencing.
Highlights
High-molecular-weight and pure DNA is crucial for high-quality results from 3rd generation DNA Analyzers and optical mapping technologies
Second generation DNA sequencing technologies, such as Illumina and Ion Torrent are beginning to become shadowed by long read length capabilities and improving accuracies of 3rd generation technologies, such as 10 × Genomics [1], Pacific Biosciences [2], and Oxford nanopore [3]
The genomic DNA (gDNA) yield from each of these species is greater than 100 micrograms of gDNA per 10 grams fresh tissue, which meets the needs for single-molecule technologies
Summary
High-molecular-weight and pure DNA is crucial for high-quality results from 3rd generation DNA Analyzers and optical mapping technologies. Second generation DNA sequencing technologies (short read), such as Illumina and Ion Torrent are beginning to become shadowed by long read length capabilities and improving accuracies of 3rd generation technologies, such as 10 × Genomics [1], Pacific Biosciences [2], and Oxford nanopore [3] These long read platforms have facilitated a shift in whole genome sequencing strategies from hybrid approaches (short + long-read) to a singular platform for de novo data collection and/or resequencing. Genomic physical mapping has seen a transition from hierarchical BAC-by-BAC approaches to optical mapping, with the primary platform being the Saphyr® [4] As these single-molecule technologies continue to evolve, the need for high purity, extremely large, in-tact genomic DNA continues to rise. This step is commonly performed by grinding tissues in the presence of liquid nitrogen; (2) Breaking the organelles to release organellar DNA, other than the nuclei, with an osmotic nuclear
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